Array assays between surface bound binding agents or probes and target molecules in solution may be used to detect the presence of particular analytes or biopolymers in the solution. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules capable of binding with target biomolecules in the solution. Such binding interactions are the basis for many of the methods and devices used in a variety of different fields, e.g., genomics (in sequencing by hybridization, SNP detection, differential gene expression analysis, identification of novel genes, gene mapping, finger printing, etc.) and proteomics.
One typical array assay method involves biopolymeric probes immobilized in an array on a substrate such as a glass substrate or the like. A solution containing target molecules (“targets”) that bind with the attached probes is placed in contact with the bound probes under conditions sufficient to promote binding of targets in the solution to the complementary probes on the substrate to produce a binding complex that is bound to the surface of the substrate. The pattern of binding by target molecules to probe features or spots on the substrate produces a pattern, i.e., a binding complex pattern, on the surface of the substrate that is detected. This detection of binding complexes provides desired information about the target biomolecules in the solution.
The binding complexes may be detected by reading or scanning the array with, for example, optical means—although other methods may also be used, as appropriate for the particular assay. For example, laser light may be used to excite fluorescent labels attached to the targets, generating a signal only in those spots on the array that have a labeled target molecule bound to a probe molecule. This pattern may then be digitally scanned for computer analysis. Such patterns can be used to generate data for biological assays such as the identification of drug targets, single-nucleotide polymorphism mapping, monitoring samples from patients to track their response to treatment, assessing the efficacy of new treatments, etc.
In array fabrication, the quantities of biopolymer available, whether by deposition of previously obtained biopolymers or by in situ synthesis, are usually very small and expensive. Additionally, sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require use of arrays with large numbers of very small, closely spaced features.
Scanning all these features can take considerable time. Over this time, it has been observed that an array may suffer degradation in the signal strength obtained toward the latter stages of a scan run. Further degradation may result if the arrayreading instrument holds multiple arrays, and takes even more time to completely scan all of the features on all of the arrays. The inventor(s) hereof recognized that microarray sensitivity to ozone is often causative of this observed effect.
Ozone has been found to attack and degrade the fluorescent dyes used in arrays—particularly Cyanine dyes, especially Cy5. Significant research has been undertaken in understanding, measuring and documenting this effect. See, e.g., Effects of Atmospheric Ozone on Microarray Data Quality by Fare, et al., Anal. Chem. 2003, Vol. 75 No. 17, 4672-4675. Indeed, it has been found that in just 5 minutes within a scanner, potential signal loss as a result of Cy5-ozone interaction can approach 1% of the original signal intensity. After an hour in the scanner, a loss of about 15% in Cy5 signal intensity may be observed While higher levels of ozone result in damage more quickly, it has been demonstrated that arrays are sensitive to levels of roughly 10 ppb or more during a typical scanning time of a microarray.
Several approaches have been used in effort to address the problem of array degradation by ozone. One regimen contemplates simply storing an array in a container (or cabinet) that is free from ozone. One may simply employ an array/slide holder that is effectively (though not necessarily hermetically) sealed.
Another approach is to limit the effect of ozone by processing an array quickly and scanning it soon thereafter. In a similar approach, another manner of avoiding ozone degradation has been to limit the time for which microarrays remain in a scanner microarray feeder queue. Yet, as alluded to above this goal may sometimes be unattainable in that an entire automated run of, for example, 48 slides (microarrays) can take 8-plus hours.
What is more, an issue of variation in scanning time is present—partly due to scheduling issues, different scan types used by an operator, speeds at which the arrays may be scanned etc.—that adds an undesirable level of uncertainty to scan results. Still further, the point at which a particular array/slide is scanned during a run introduces variability in the degree of signal measured in accordance to array position within the scanning queue (i.e., timing variability is introduced by way of which arrays are scanned sooner, and which arrays are scanned later in the sequence). As long as ozone attack persists, time represents a statistically significant variable
Another treatment of the symptoms of array/dye degradation over time has been to provide an array with a protective chemical coating to reduce degradation rates. One such chemical coating is known as the DyeSaver compound sold by Genisphere. Yet, even this approach has potential drawbacks. Specifically, it offers no protection during array drying and is not effective with certain types of oligonucleotide arrays.
On the other hand, array degradation can be prevented if the slides are stored in Nitrogen (or another neutral atmosphere). Though providing an inert atmosphere is not an option, on a larger scale it has also been appreciated that an entire working environment can be scrubbed of harmful gasses. This goal has been accomplished by employing a clean-room type filter system for ozone in the scanner and/or array processing environment—as has been previously done in microchip fabrications facilities. However, the hardware investment, requirement for HVAC installation, and/or energy expenditures can be significant, setup for such a system can be time-consuming and inconvenient to implement, and even then, it will not be 100% effective. Related approaches that have been tried or suggested are to carry out array processing and/or scanning under an ozone-filtered hood or customized “bio-bubble”. Even so, these other approaches can involve significant cost or inconvenience.
Instead, the present invention offers a variety of alternative solutions that will be preferred for their avoidance of undue trouble and cost—as in filtering an entire room/building of ozone. Stated otherwise, systems according to the present invention may offer benefits or gains in terms of convenience, and efficiency in protecting arrays from unwanted chemical interaction. As important (or more so), gains may be observed in terms of scanning efficacy by virtue of features of the present invention.